ThisisaclassicalmethodofmethylationanalysisbasedonthepropertyofsomerestrictionenzymestobeunabletocutmethylatedDNA.SinceineukaryoticDNA(oractuallyinmammalianDNA)onlycytosineinCGcontextcanbemethylated,therestrictionenzymeswithCGsequenceswithintheirrestrictionsitescomeinquestion.TwoclassicalenzymepairsareHpaII-MspI(CCGG)andSmaI-XmaI(CCCGGG).SincethesecondrecognitionsequenceismuchmorerarethanthefirstoneIwillconcentrateontheHpaII-MspIpair.BothenzymesrecognizeCCGGsequence,howeverHpaIIisunabletocutDNAwhentheinternalcytosineismethylated.ThispropertymakesHpaII-MspIpairtoavaluabletoolforrapidmethylationanalysis.

Thismethodhassomeweakpoints.FirstisthatnotallCGarelocatedwithinCCGGsequences,thatmeansmanypotentialmethylationsiteswillbeoverlooked.AnotherproblemisanecessityofaSouthernblothybridisationwhichisnotuncomplicated.Howeveritisstillagoodmethodtogetafirstideaifthemethylationisinvolvedinthephenomenonyouobserve.

Beforeyouproceedwithamethylationanalysisyouhavetoanalyseyoursequenceverycarefully.Ifyou"lljusttakegenomicDNA,digestitwithHpaII,blotandhybridisewithselectedprobetheresultwillbeverycomplicated.With200bpprobeyoucanobtainmyriadofbandswhichyouwillnotbeabletointerpret.ThereforefirstofalldefinetheregionofinterestflankedwithrestrictionsitesforCGmethylationinsensitiveenzymes(BamHIforexample),andcontainingnotmorethan5-6sitesforHpaII.TheprobeusedforSouthernblothybridisationshouldbelocatedwithinthisregionandcoveritcompletelyorpartially(seethefigure).

EveninthiscasewhenyouwillusetheprobeAthenumberoffragmentswillberelativelyhigh(trytocountthemifyoumakecompletedigestionwithBamHIandthen-incompletewithHpaII).ProbeBwillgiveyoulesscomplexpicture,butactuallythesameinformation.Anotherproblemisthesizeofthefragmentsyouusuallyobtain.IfyouhaveaCGrichregion,thenHpaIIrestrictionfragmentswillbeintherange100-500bp.Forthisfragmentrangeyouwillneed1.2%agaroseandsuchgelsarerelativelydifficulttoblot.Howeverthismethodseemstobesimplerandfastertoestablishthanbisulphitetreatment.

Protocol

1. IsolategenomicDNAwithanymethodthatproduceDNAofqualitysufficientforrestrictiondigestion.
2. Digest10µgofDNAovernightwithafirstenzyme,cuttingoutthefragmentofinterestincombinationwithHpaII,andanother10µgwithfirstenzymeincombinationwithMspI.Reactionvolumeshouldbeabout50µl.
3. Inactivatetheenzymesbyheating(lookinthedatasheetofthemanufacturerforinactivationprotocol).
4. Reducethesamplevolumetoabout20-30µlinthevacuumconcentrator.DoNOTdrytheDNAyouwillnotbeabletodissolveitagain!
5. AddrequiredvolumeofloADIngbufferandloadthesamplesontothe1.2%agarose/TAEgel.Usuallya20cmlonggelisenough.Thewellsinthegelshouldbe5-7mmwideforthebetterqualityofthebands.
6. Runthegelat1-2V/cm.
7. PerformstandardSouthernblottingandhybridisation.
8. ThebandsinMspIdigestedsamplelaneshouldrepresentacompletedigestionofyourDNAwithfirstenzymeandMspI,andshouldserveasacontrolforthedigestioncompletenessandtheabsenceofmutations.
9. DeterminethesizesofthebandsinHpaIIdigestedsamplesandfindthecorrespondingrestrictionsitesinthesequence.

Donotexpecttoobtainaclearcutresults.Usuallycelllinesandespeciallytissuesarepolyclonalandcontainmixedmethylationpatterns,thereforeyourbandpatternwillbealsocomplex.